Cellular mechanosensing is a fundamental component of a number of human diseases including osteoporosis, atherosclerosis, osteoarthritis, and cancer. New therapeutic targets are in critical need, but pharmacologically manipulating the cellular mechanosensory apparatus remains essentially unexplored. Primary cilia are solitary cellular organelles that form signaling microdomains and act as extracellular sensors in many different tissues and contexts. Recent in vitro and in vivo data from our lab (in the parent grant to this application) has established their role as mechanosensors in osteocytes. Others have shown that primary cilia act as mechanosensors in the cells of cartilage, arterial endothelia, kidney epithelia, metastases, and even the embryonic node. In order to develop breakthrough therapeutics, there is an unmet need to identify agents that affect the mechanosensitivity of primary cilia. The long-term goal of this project is to identify strategies t manipulate cellular mechanosensitivity. The overall objective of this application is to identify small molecules or classes thereof that enhance responsiveness. However, traditional mechanobiology (e.g. applying flow or stretch and assessing gene or protein expression) cannot be quantified in a high-throughput format. We will overcome this limitation by using primary cilia length as a surrogate measure, since we have linked it to mechanosensing and it is easily quantified optically. Thus, our central hypothesis is that increasing ciliary length will increase cellular responsiveness. Our rationale, supported by preliminary data, is that membrane strain is increased with ciliary length, which will enhance transmembrane ion channel activity, similar to chemical agonists. We will test our central hypothesis with a new integrated experimental/theoretical collaboration between Christopher Jacobs and Brent Stockwell. Experimentally we will utilize a high-content small molecule screen and validate candidates by measuring mechanosensitivity (Specific Aim 1). We will also use computational chemistry to optimize receptor-ligand interactions involved in the Ca2+/cAMP signaling cascade, which we have shown to be mechanically induced within the ciliary microdomain (Specific Aim 2). These two highly synergistic strategies, enabled by advances in modern chemistry, feed into one another, but have yet to be applied to mechanosensing.